Rapid Proteolysis Research Articles

Sodium alginate/polyacrylic acid hydrogel composite, potential carrier for fibroblast growth factor1 (FGF1) delivery.

Fibroblast growth factor1 is a powerful signaling molecule that plays a critical role in injury repair of diverse tissue by stimulating cell growth and angiogenesis. FGF1 has significant role in the cell fate and regulating inflammation with short half-life and poor in vivo stability. The encapsulation of the growth factor in the hydrogel led to peptide protect from the degradation and/or immune recognition and enable controlled drug delivery over a longer period of time. The aim of this study is to develop and evaluate a hydrogel carrier with adjustable release rate while maintaining bioactivity of FGF1. Here we describe an optimal ratio of sodium alginate and polyacrylic acid without additional cross linker containing FGF1 with the potential of sustained release to be used as a therapeutic agent. The carrier was characterized by FTIR, contact angel and swelling ratio and then variety of FGF1 concentration tested to find the optimum release. The activity of FGF1 after release from the hydrogel was confirmed by ELISA and Western blot. This hydrogel is able to deliver growth factors by restricting the essential proteins within the matrix to prevent rapid proteolysis and explosive release and is therefore widely applicable.

Versatile Fabrication of Biocompatible Antimicrobial Materials Enabled by Cationic Peptide Bundles.

Antimicrobial peptides (AMPs) are expected to be an alternative promising solution to the global public health problem of antibiotic resistance due to their unique antimicrobial mechanism. However, extensive efforts are still needed to improve the shortcomings of traditional AMPs, such as rapid proteolysis, hemolysis, slow response, toxicity, etc., by exploring AMP-based new antimicrobial strategies. Here, we develop cationic peptide bundles into novel antimicrobial architectures that can rapidly kill multiple types of bacteria including drug-resistant bacteria. Remarkably, cationic peptide bundles can be used as polymerization units to cross-link with other polymers through simple two-component polymerization to produce diverse antimicrobial materials. For the proof of concept, three materials were fabricated and investigated, including an antimicrobial hydrogel that can significantly accelerate the healing of infected wounds, a multifunctional antimicrobial bioadhesive that shows promise in antimicrobial coatings for medical devices, and a photo-cross-linked antimicrobial gelatin hydrogel with broad application potential. The integration of antimicrobial units into the materials' backbone endows their biocompatibility. Cationic peptide bundles not only represent a new antimicrobial strategy but also provide a versatile and promising processing method to create diversified, multifunctional, and biocompatible antimicrobial materials.

Hydrogel-Based Growth Factor Delivery Platforms: Strategies and Recent Advances.

Growth factors play a crucial role in regulating a broad variety of biological processes and are regarded as powerful therapeutic agents in tissue engineering and regenerative medicine in the past decades. However, their application is limited by their short half-lives and potential side effects in physiological environments. Hydrogels are identified as having the promising potential to prolong the half-lives of growth factors and mitigate their adverse effects by restricting them within the matrix to reduce their rapid proteolysis, burst release, and unwanted diffusion. This review discusses recent progress in the development of growth factor-containing hydrogels for various biomedical applications, including wound healing, brain tissue repair, cartilage and bone regeneration, and spinal cord injury repair. In addition, the review introduces strategies for optimizing growth factor release including affinity-based delivery, carrier-assisted delivery, stimuli-responsive delivery, spatial structure-based delivery, and cellular system-based delivery. Finally, the review presents current limitations and future research directions for growth factor-delivering hydrogels.

Insights on the conformation and appropriate drug-target sites on retinal IMPDH1 using the 604-aa isoform lacking the C-terminal extension.

Retinitis pigmentosa (RP) accounts for 2 percent of global cases of blindness. The RP10 form of the disease results from mutations in isoform 1 of inosine 5'-monophosphate dehydrogenase (IMPDH1), the rate-limiting enzyme in the de novo purine nucleotide synthesis pathway. Retinal photoreceptors contain specific isoforms of IMPDH1 characterized by terminal extensions. Considering previously reported significantly varied kinetics among retinal isoforms, the current research aimed to investigate possible structural explanations and suitable functional sites for the pharmaceutical targeting of IMPDH1 in RP. A recombinant 604-aa IMPDH1 isoform lacking the carboxyl-terminal peptide was produced and underwent proteolytic digestion with α-chymotrypsin. Dimer models of wild type and engineered 604-aa isoform were subjected to molecular dynamics simulation. The IMPDH1 retinal isoform lacking C-terminal peptide was shown to tend to have more rapid proteolysis (~16% digestion in the first two minutes). Our computational data predicted the potential of the amino-terminal peptide to induce spontaneous inhibition of IMPDH1 by forming a novel helix in a GTP binding site. On the other hand, the C-terminal peptide might block the probable inhibitory role of the N-terminal extension. According to the findings, augmenting IMPDH1 activity by suppressing its filamentation is suggested as a suitable strategy to compensate for its disrupted activity in RP. This needs specific small molecule inhibitors to target the filament assembly interface of the enzyme.

A novel device for swift and efficient CD44 protein digestion of pipette tips in human serum

For molecular diagnostics in modern biomedical research, electrospray ionisation mass spectrometry (ESI-MS) based on proteome profiling is important. Now a days, sample preparation such as proteolysis and protein extraction remain incredibly challenging and inefficient. Recent sample-preparation methods based on micro tips show promising results toward the aim “a proteome in an hour”. Proteolysis at the tip, is still infrequently observed and does not represent the processing of complex bio-samples. In this study, we outline a unique technique for detecting and extracting human serum CD44 biomarkers by ligand–protein interactions. This method employs macropores silica particles (MPSP) or (MOSF) modified with hyaluronic acid (HA). In order to assist in the profile of the human serum proteome, we limitations of immunoassays for rapid and multimodal proteolysis. For effective in situ proteolysis, in micropipette tips, MPSP were designed as nanoreactors with variable pore size and surface chemistry. In MS-based bottom-up proteome analysis, the device as-built demonstrated favourable sensitivity (LOD of 0.304 ± 0.007 ng/mL and LOQ of 0.973 ± 0.054 ng/mL), selectivity, durability (at −20 °C for 2 months), reuse (at least 10 times), and minimal memory impact. In addition, we examined into specific surface chemistries of nanoparticles for the absorption of proteins in serum and profiled the HA-binding serum proteome, setting a new preliminary benchmark for future databases. Our study not only helped establish a new platform for extracting/detection of CD44 and identifying the HA-binding proteome, but it also offered design recommendations for ligand affinity-based techniques for the antibody-free study of serum biomarkers with a view towards diagnostic applications.

Peptide Transporter OsNPF8.1 Contributes to Sustainable Growth under Salt and Drought Stresses, and Grain Yield under Nitrogen Deficiency in Rice

Peptide transport is important for plant tissues where rapid proteolysis occurs, especially during germination and senescence, to enhance redistribution of organic nitrogen (N). However, the biological role of peptide transporters is poorly investigated in rice. We characterized the function of the peptide transporter OsNPF8.1 of rice nitrate transporter 1/peptide transporter family (NPF). Ectopic expression of OsNPF8.1 in yeast revealed that OsNPF8.1 encoded a high-affinity di-/tri-peptide transporter, and the osnpf8.1 mutants had a lower uptake rate of the fluorescent-labelled dipeptide c in leaves of rice seedlings. Histochemical assays showed that OsNPF8.1 was highly expressed in mesophyll cells and vascular parenchyma cells, but not detected in root hairs and epidermises. Expression of OsNPF8.1 was induced by N deficiency, drought, NaCl and abscisic acid, and kept at a high level in senescing leaves. Under N deficiency conditions, compared with the wild type Zhonghua 11, the osnpf8.1 mutants grew slower at the seedling stage, and had lower grain yield and lower N content in the grains. In contrast, OsNPF8.1-over-expressing rice (OsNPF8.1-OE) grew faster at the seedling stage and had a higher grain yield. The osnpf8.1 seedlings were less tolerant to salt and drought stresses. These results suggested that stress-induced organic N transportation mediated by OsNPF8.1 might contribute to balance plant growth and tolerate to salt/drought stress and N-deficiency.

Design of Volumetric Nanolayers via Rapid Proteolysis of Silk Fibroin for Tissue Engineering.

Various methods have been studied to make a regenerated silk fibroin solution. However, most of them take too much time and effort to liquefy. Here, we report that a regenerated silk fibroin solution could be prepared within seconds through acid proteolysis for the first time. The solubilized fibroin could be applied to advanced tissue engineering. Our method shortened the production time to one day (more than 10 times) compared to the general fibroin solution preparation method. It was confirmed that the initial protein affinity nearly doubled from 0.028 to 0.076 μg·mm-2 in FF(ac) compared to FF(aq). A fibroin nanofiber layer having a volumetric hierarchical structure was prepared by electrospinning an acid-proteolyzed fibroin solution, followed by gas foaming. In vitro results of cell adhesion and proliferation capacity of the gas-foamed scaffold were not significantly different compared to the two-dimensional (2D) fibroin nanofiber membrane, overcoming the limitations of volumetric nanofiber scaffolds. We are confident that our research will greatly contribute to the development of regenerative engineering using other proteins.

Detection of Damage-Activated Metacaspase Activity by Western Blot in Plants.

Metacaspases are cysteine proteases that are present in plants, protists, fungi, and bacteria. Previously, we found that physical damage, e.g., pinching with forceps or grinding on liquid nitrogen of plant tissues, activates Arabidopsis thaliana METACASPASE 4 (AtMCA4). AtMCA4 subsequently cleaves PROPEP1, the precursor pro-protein of the plant elicitor peptide 1 (Pep1). Here, we describe a protein extraction method to detect activation of AtMCA4 by Western blot with antibodies against endogenous AtMCA4 and a PROPEP1-YFP fusion protein. It is important to (1) keep plant tissues at all times on liquid nitrogen prior to protein extraction, and (2) denature the protein lysate as fast as possible, as metacaspase activation ensues quasi immediately because of tissue damage inherent to protein extraction. In theory, this method can serve to detect damage-induced alterations of any protein-of-interest in any organism for which antibodies or fusion proteins are available, and hence, will greatly aid the study of rapid damage-activated proteolysis in the future.

Rapid proteolysis of gluten-derived immunogenic peptides in bread by actinidin in a combined in vivo and in vitro oro-gastrointestinal digestion model.

This study aimed to determine the ability of actinidin, a cysteine protease in green kiwifruit (Actinidia deliciosa), to hydrolyse wheat proteins and gluten-derived immunogenic peptides from a commonly consumed food matrix (bread) using a combined in vivo and in vitro oro-gastrointestinal tract (GIT) model. A chewed and spat composite bolus of bread was in vitro digested with or without purified actinidin using a human gastric simulator (HGS). Gastric digestion was conducted for 150 min with gastric emptying occurring at different time points. Emptied samples were immediately digested under simulated small intestinal conditions. Gastric and small intestinal aliquots were collected to quantify peptide profiles and nine marker immunogenic peptides (by untargeted and targeted mass spectrometry, respectively), R5 epitopes (by monoclonal antibody-based competition assay), and free amino groups released by digestion (by the o-phthaldialdehyde method). There was a significant effect (P < 0.05) of actinidin and digestion time on the hydrolysis of wheat proteins and the amount of gluten R5 epitopes of that material emptying the HGS. Actinidin accelerated 1.2-fold the gastric hydrolysis of wheat proteins during the first 20 min of digestion, which was reflected in a faster (5.5 μg min-1) reduction in the evolution of R5 epitopes. Actinidin accelerated (P < 0.05) the rate of disappearance of most of the immunogenic marker peptides. For example, in the first 20 min of small intestinal digestion, the 33-mer peptide decreased (P < 0.05) 2-fold faster (0.25 vs. 0.12 μg g-1 of bread per min) in the presence of actinidin than in the control. Untargeted peptidomics showed actinidin decreased the amounts of known immunogenic peptides in the simulated small intestinal digestion. These findings demonstrated that actinidin accelerates the hydrolysis of wheat proteins and known gluten immunogenic peptides in a commonly consumed food matrix (bread) in a combined in vivo and in vitro oro-GIT digestion model.

Controllable Synthesis of Hollow Microtubular Covalent Organic Frameworks as an Enzyme-Immobilized Platform for Enhancing Catalytic Activity.

Despite great achievement that has been made in the synthesis of covalent organic frameworks (COFs), precise construction of COFs with well-defined nano/microstructures poses a rigorous challenge. Herein, we introduce a simple template-free strategy for controllable synthesis of hollow microtubular COFs. The obtained COFs show a spontaneous morphology transformation from a microfiber to a hollow microtubular structure when the concentrations of catalytic acid are regulated elaborately. Furthermore, the as-prepared COFs exhibit high crystallinity, well-defined hollow tubular morphology, and high surface areas (∼2600 m2/g). Taking the advantages of the unique morphological structure, the hollow microtubular COFs can serve as an ideal host material for enzymes. The resultant biocomposites show high catalytic performance and can be successfully applied to rapid and high-efficiency proteolysis of proteins. This work blazes a trail for controllable synthesis of the hollow microtubular COFs through a template-free process and expands the application of COFs as a promising platform for enzyme immobilization.

The combined effect of permeation enhancement and proteolysis inhibition on the systemic exposure of orally administrated peptides: Salcaprozate sodium, soybean trypsin inhibitor, and teriparatide study in pigs

Oral delivery of peptides and proteins is hindered by their rapid proteolysis in the gastrointestinal tract and their inability to permeate biological membranes. Various drug delivery approaches are being investigated and implemented to overcome these obstacles. In the discussed study conducted in pigs, an investigation was undertaken to assess the effect of combination of a permeation enhancer – salcaprozate sodium, and a proteolysis inhibitor – soybean trypsin inhibitor, on the systemic exposure of the peptide teriparatide, following intraduodenal administration. Results demonstrate that this combination achieves significantly higher Cmax and AUC (~10- and ~20-fold respectively) compared to each of these methodologies on their own. It was thus concluded that an appropriate combination of different technological approaches may considerably contribute to an efficient oral delivery of biological macromolecules.

Subsynaptic positioning of AMPARs by LRRTM2 controls synaptic strength.

Recent evidence suggests that nano-organization of proteins within synapses may control the strength of communication between neurons in the brain. The unique subsynaptic distribution of glutamate receptors, which cluster in nanoalignment with presynaptic sites of glutamate release, supports this hypothesis. However, testing it has been difficult because mechanisms controlling subsynaptic organization remain unknown. Reasoning that transcellular interactions could position AMPA receptors (AMPARs), we targeted a key transsynaptic adhesion molecule implicated in controlling AMPAR number, LRRTM2, using engineered, rapid proteolysis. Severing the LRRTM2 extracellular domain led quickly to nanoscale declustering of AMPARs away from release sites, not prompting their escape from synapses until much later. This rapid remodeling of AMPAR position produced significant deficits in evoked, but not spontaneous, postsynaptic receptor activation. These results dissociate receptor numbers from their nanopositioning in determination of synaptic function and support the novel concept that adhesion molecules acutely position receptors to dynamically control synaptic strength.

Pharmacokinetics of Subcutaneously Administered Simoctocog Alfa with Von Willebrand Factor Fragment Candidates in a Minipig Model

Introduction/Objective: The use of the subcutaneous (SC) administration route for hemophilia A therapy is hindered by the very low bioavailability of Factor VIII (FVIII) after SC administration. von Willebrand factor (VWF), the natural chaperone for FVIII, protects FVIII from rapid proteolysis, clearance, binding to phospholipids and uptake by macrophages. These mechanisms most likely contribute to the entrapment and degradation of FVIII in the SC tissue. Therefore, the use of VWF or VWF fragments represents a promising strategy in enabling FVIII to enter the circulation after SC administration. To evaluate this concept, we tested two VWF fragments in a minipig model.Materials/Methods: Two VWF fragments, O™CTA12 and OCTA13, with a C-terminal Strep-tag™ were expressed in a HEK293 cell line and purified by Strep-Tactin™ affinity chromatography. VWF fragment OCTA12 is a dimer containing D'D3 domains engineered to increase the glycosylation level. VWF fragment OCTA13 is a dimer containing D'D3-A1-A2-A3 domains. Both fragments were biochemically characterized by SDS-PAGE, FVIII binding and the ability to inhibit the interaction between FVIII and phospholipids. In addition, the ability of the VWF fragments to interfere with full-length VWF (flVWF) for mediation of platelet adhesion to collagen type III in a flow chamber was examined. Aachener minipigs (three animals per group) were administered with 100 IU/kg BW recombinant human FVIII (rhFVIII; simoctocog alfa) SC either alone or in combination with OCTA12 or OCTA13. As a control the same dose of rhFVIII was administered IV. Blood was taken prior to and 0.5, 1, 2, 4, 8, 24, 32, 48, 72, 96 and 120 h after each administration. The human FVIII antigen concentration and OCTA12 or OCTA13 concentrations were tested in each sample.Results:As confirmed by SDS-PAGE, both VWF fragments were expressed as dimers with minor monomer content. The propeptide was almost entirely cleaved off. The FVIII binding affinity (KD, mean of three measurements) was 0.93 nM for OCTA12 and 1.81 nM for OCTA13. About 90% inhibition of interaction between FVIII and phospholipid monolayer could be achieved for 6-fold molar excess of each fragment. Consequently, this molar ratio was used for the animal studies. No inhibition of full-length VWF activity by OCTA12 or OCTA13 could be observed in the flow chamber assay. The FVIII peak concentrations were as follows: 2 IU/ml at 1 h time point for rhFVIII alone administered IV; 0.02 IU/ml at 8 h time point for rhFVIII alone administered SC; 0.23 IU/ml at 8 h time point for rhFVIII complexed with OCTA12 administered SC. FVIII bioavailability was determined as the area under the curve (AUC) of FVIII:Ag, and the AUC after IV administration was defined as 100%. Bioavailability of FVIII alone after SC administration was 2%. The co-administration of rhFVIII with OCTA12 and OCTA13 increased the bioavailability of FVIII to 41% and 32.9%, respectively. Co-administration of rhFVIII with OCTA12 resulted in 2.3-fold longer time to 1% FVIII trough level compared with IV administration of FVIII at the same dose. Peak levels of 1.17 nM and 0.8 nM VWF fragment could be measured at 8 h for OCTA12 and OCTA13, respectively. At 120 h, 0.14 nM of OCTA13 and a still very high concentration of 0.83 nM of OCTA12 antigen could be measured, indicating a long half-life and/or slow release into the circulation.Conclusions:The two novel VWF fragments OCTA12 and OCTA13 significantly increased the bioavailability of FVIII after SC administration. High levels of VWF fragment OCTA12 could be detected over the entire sampling period, indicating a very long circulatory half-life and/or slow sustainable release of the fragment into the circulation. Importantly, the slow release and/or prolonged circulatory half-life of OCTA12 contributed to significantly prolonged FVIII antigen levels in the circulation after SC administration. In summary, the use of OCTA12 as a FVIII chaperone for SC administration represents a promising strategy to improve hemophilia A therapy in terms of application route and dosing frequency. DisclosuresSolecka-Witulska:Octapharma Biopharmaceuticals GmbH: Employment. Wuschko:Octapharma Pharmazeutika Produktionsges.m.b.H.: Employment. Kannicht:Octapharma Biopharmaceuticals GmbH: Employment.

FBX4 mediates rapid cyclin D1 proteolysis upon DNA damage in immortalized esophageal epithelial cells

It has been implied that deregulation of cyclin D1 turnover under stresses can facilitate genomic instability and trigger tumorigenesis. Much focus has been placed on identifying the E3 ligases responsible for mediating cyclin D1 degradation. However, the findings were quite controversial and cell type-dependent. Little is known about how cyclin D1 is regulated in precancerous cells upon DNA damage and which E3 ligases mediate the effects. Here we found cyclin D1 reduction is an early response to DNA damage in immortalized esophageal epithelial cells, with expression dropping to a low level within 1 h after γ-irradiation. Comparison of temporal expression of cyclin D1 upon DNA damage between immortalized NE083-hTERT and NE083-E6E7, the latter being p53/p21-defective, showed that DNA damage-induced rapid cyclin D1 reduction was p53-independent and occurred before p21 accumulation. Overexpression of cyclin D1 in NE083-E6E7 cells could attenuate G0/G1 cell cycle arrest at 1 h after irradiation. Furthermore, rapid reduction of cyclin D1 upon DNA damage was attributed to proteasomal degradation, as evidenced by data showing that proteasomal inhibition by MG132 blocked cyclin D1 reduction while cycloheximide facilitated it. Inhibition of ATM activation and knockdown of E3 ligase adaptor FBX4 reversed cyclin D1 turnover in immortalized NE083-hTERT cells. Further study showed that knockdown of FBX4 facilitated DNA breaks, as indicated by an increase in γ-H2AX foci in esophageal cancer cells. Taken together, the results substantiated a pivotal role of ATM and FBX4 in cyclin D1 proteolysis upon DNA damage in precancerous esophageal epithelial cells, implying that deregulation of the process may contribute to carcinogenesis of esophageal squamous cell carcinoma.

Snx4-assisted vacuolar targeting of transcription factors defines a new autophagy pathway for controlling ATG expression

ABSTRACT Autophagy, in part, is controlled by the repression and activation of autophagy-related (ATG) genes. Here, we describe a new selective autophagy pathway that targets functional transcriptional regulators to control their activity. This pathway is activated in response to nitrogen starvation and recycles transcriptional activators (Msn2 and Rim15) and a repressor (Ssn2/Med13) of ATG expression. Further analysis of Ssn2/Med13 vacuolar proteolysis revealed that this pathway utilizes the core autophagic machinery. However, it is independent of known nucleophagy mechanisms, receptor proteins, and the scaffold protein Atg11. Instead, Ssn2/Med13 exits the nucleus through the nuclear pore complex (NPC) and associates with the cytoplasmic nucleoporin Gle1, a member of the RNA remodeling complex. Dbp5 and Nup159, that act in concert with Gle1, are also required for Ssn2/Med13 clearance. Ssn2/Med13 is retrieved from the nuclear periphery and degraded by Atg17-initiated phagophores anchored to the vacuole. Efficient transfer to phagophores depends on the sorting nexin heterodimer Snx4/Atg24-Atg20, which binds to Atg17, and relocates to the perinucleus following nitrogen starvation. To conclude, this pathway defines a previously undescribed autophagy mechanism that targets select transcriptional regulators for rapid vacuolar proteolysis, utilizing the RNA remodeling complex, the sorting nexin heterodimer Snx4-Atg20, Atg17, and the core autophagic machinery. It is physiologically relevant as this Snx4-assisted vacuolar targeting pathway permits cells to fine-tune the autophagic response by controlling the turnover of both positive and negative regulators of ATG transcription. Abbreviations: AIM: Atg8 interacting motif; ATG: autophagy-related; CKM: CDK8 kinase module; IDR: intrinsically disordered region; IP6: phosphoinositide inositol hexaphosphate; NPC: nuclear pore complex; PAS: phagophore assembly site; UPS: ubiquitin-proteasomal system

Characterizing Native and Hydrocarbon-Stapled Enfuvirtide Conformations with Ion Mobility Mass Spectrometry and Hydrogen-Deuterium Exchange.

The number of approved peptide therapeutics, as well as those in development, has been increasing in recent years. Frequently, the biological activity of such peptides is elicited through the adoption of secondary structural elements upon interaction with their cellular target. However, many therapeutic peptides are unstructured in solution and accordingly exhibit a poor bioavailability due to rapid proteolysis in vivo. To combat this degradation, numerous naturally occurring peptides with therapeutic properties contain stabilizing features, such as N-to-C cyclization or disulfide bonds. Recently, hydrocarbon stapling via non-native amino acid substitution followed by ring-closing metathesis has been shown to induce a dramatic stabilization of α-helical peptides. Identifying the ideal staple location along the peptide backbone is a critical developmental step, and methods to streamline this optimization are needed. Mass spectrometry-based methods such as ion mobility (IM) and hydrogen-deuterium exchange (HDX) can detect multiple discrete peptide conformations, a significant advantage over bulk spectroscopic techniques. In this study we use IM-MS and HDX-MS to demonstrate that the native 36-residue enfuvirtide peptide is highly dynamic in solution and the conformational ensemble populated by stabilized constructs depends heavily on the staple location. Further, our measurements yielded results that correlate well with the average α-helical content measured by circular dichroism. The MS-based approaches described herein represent sensitive and potentially high-throughput methods for characterizing and identifying optimally stapled peptides.

A N-Terminus Domain Determines Amelogenin's Stability to Guide the Development of Mouse Enamel Matrix.

Amelogenins, the principal proteins in the developing enamel microenvironment, self-assemble into supramolecular structures to govern the remodeling of a proteinaceous organic matrix into longitudinally ordered hydroxyapatite nanocrystal arrays. Extensive in vitro studies using purified native or recombinant proteins have revealed the potential of N-terminal amelogenin on protein self-assembly and its ability to guide the mineral deposition. We have previously identified a 14-aa domain (P2) of N-terminal amelogenin that can self-assemble into amyloid-like fibrils in vitro. Here, we investigated how this domain affects the ability of amelogenin self-assembling and stability of enamel matrix protein scaffolding in an in vivo animal model. Mice harboring mutant amelogenin lacking P2 domain had a hypoplastic, hypomineralized, and aprismatic enamel. In vitro, the mutant recombinant amelogenin without P2 had a reduced tendency to self-assemble and was prone to accelerated hydrolysis by MMP20, the prevailing metalloproteinase in early developing enamel matrix. A reduced amount of amelogenins and a lack of elongated fibrous assemblies in the development enamel matrix of mutant mice were evident compared with that in the wild-type mouse enamel matrix. Our study is the first to demonstrate that a subdomain (P2) at the N-terminus of amelogenin controls amelogenin's assembly into a transient protein scaffold that resists rapid proteolysis during enamel development in an animal model. Understanding the building blocks of fibrous scaffold that guides the longitudinal growth of hydroxyapatites in enamel matrix sheds light on protein-mediated enamel bioengineering. © 2021 American Society for Bone and Mineral Research (ASBMR).

Equilibrium between nascent and parental MCM proteins protects replicating genomes.

Minichromosome maintenance proteins (MCMs) are DNA-dependent ATPases that bind to replication origins and license them to support a single round of DNA replication. A large excess of MCM2-7 assembles on chromatin in G1 phase as pre-replication complexes (pre-RCs), of which only a fraction become the productive CDC45-MCM-GINS (CMG) helicases that are required for genome duplication1-4. It remains unclear why cells generate this surplus of MCMs, how they manage to sustain it across multiple generations, and why even a mild reduction in the MCM pool compromises the integrity of replicating genomes5,6. Here we show that, for daughter cells to sustain error-free DNA replication, their mother cells build up a nuclear pool of MCMs both by recycling chromatin-bound (parental) MCMs and by synthesizing new (nascent) MCMs. Although all MCMs can form pre-RCs, it is the parental pool that is inherently stable and preferentially matures into CMGs. By contrast, nascent MCM3-7 (but not MCM2) undergo rapid proteolysis in the cytoplasm, and their stabilization and nuclear translocation require interaction with minichromosome-maintenance complex-binding protein (MCMBP), a distant MCM paralogue7,8. By chaperoning nascent MCMs, MCMBP safeguards replicating genomes by increasing chromatin coverage with pre-RCs that do not participate on replication origins but adjust the pace of replisome movement to minimize errors during DNA replication. Consequently, although the paucity of pre-RCs in MCMBP-deficient cells does not alter DNA synthesis overall, it increases the speed and asymmetry of individual replisomes, which leads to DNA damage. The surplus of MCMs therefore increases the robustness of genome duplication by restraining the speed at which eukaryotic cells replicate their DNA. Alterations in physiological fork speed might thus explain why even a minor reduction in MCM levels destabilizes the genome and predisposes to increased incidence of tumour formation.

Ordered dephosphorylation initiated by the selective proteolysis of cyclin B drives mitotic exit.

APC/C-mediated proteolysis of cyclin B and securin promotes anaphase entry, inactivating CDK1 and permitting chromosome segregation, respectively. Reduction of CDK1 activity relieves inhibition of the CDK1-counteracting phosphatases PP1 and PP2A-B55, allowing wide-spread dephosphorylation of substrates. Meanwhile, continued APC/C activity promotes proteolysis of other mitotic regulators. Together, these activities orchestrate a complex series of events during mitotic exit. However, the relative importance of regulated proteolysis and dephosphorylation in dictating the order and timing of these events remains unclear. Using high temporal-resolution proteomics, we compare the relative extent of proteolysis and protein dephosphorylation. This reveals highly-selective rapid proteolysis of cyclin B, securin and geminin at the metaphase-anaphase transition, followed by slow proteolysis of other substrates. Dephosphorylation requires APC/C-dependent destruction of cyclin B and was resolved into PP1-dependent categories with unique sequence motifs. We conclude that dephosphorylation initiated by selective proteolysis of cyclin B drives the bulk of changes observed during mitotic exit.

Protein properties of UF Teleme produced from various types of milk

In this study, white cheese Teleme samples were produced from cow's, sheep's and goat's milk using UF and the traditional methods. In both methods, milk was divided into three portions and heated to 68°C/20 min, 72°C/5 min and 80°C/1 min., respectively. Each sample tested for three different pasteurization norms was divided into two portions. The calf rennet (CR) was added to the first portion and microbial rennet (MR) was added to the second portion. The teleme samples were prepared using 2 processing methods, 3 pasteurization norms,and 2 enzyme types. Besides total nitrogen and water-soluble nitrogen analyses, SDS-PAGE was applied to the samples to determine free protein breakdowns. Total nitrogen and water-soluble nitrogen values were higher in Teleme samples treated with microbial rennet than those with calf rennet in both UF and traditional Teleme samples. In the first day a rapid proteolysis and fraction product (casein fraction) just under the β-casein were determined in Teleme samples produced from milk with microbial rennet at 68°C and 72°C in SDS-PAGE. Data on water-soluble nitrogen fractions confirmed electrophoretic profiles.